Functional investigations of the human voltage-dependent anion channel 1 by solid-state NMR spectroscopy
Final Report Abstract
The original research objectives included in the fellowship application contained several sub-objectives, however, only a fraction of tasks was successfully achieved so far. Progress was mostly stunted by technical limitations at the time that were resolved very recently. The two main achievements on the hVDAC1 project are: 1) A new expression, purification, and crystallization protocol was established that was determined to be very robust. This new procedure is now used exclusively in our lab. 2) Very recently, spinning at 111 kHz MAS frequency in a 0.7 mm rotor became available. We collaborated with Guido Pintacuda (CNRS Lyon, France) in order to record hVDAC1 spectra of fully protonated samples at 110 kHz MAS at 800MHz. Due to the challenges of the hVDAC1 project, my main focus shifted towards solid-state studies of fibrillar systems, particularly amyloid beta 42 (Aβ42). These studies were significantly more successful and yielded a total of three publications on Aβ42. The three main achievements on the Aβ42 project are: 1) Amyloid-β (Aβ) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein (APP). In 2016, we published an atomic resolution structure of a monomorphic form of AβM01-42 amyloid fibrils derived from over 500 13C-13C, 13C-15N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. 2) The role of the N-terminal methionine on the structure of monomorphic Aβ42 was determined. The most detailed and reproducible insights into structure and kinetics have been achieved using Aβ peptides produced by recombinant expression, which results in an additional methionine at the N-terminus. The impact of the N-terminal methionine on the aggregation pathways and structure was unclear, therefore, we have developed a protocol to produce recombinant Aβ1-42, sans the N-terminal methionine, using an N-terminal SUMO-Aβ1-42 fusion protein. Aβ1-42 and AβM01-42 aggregate with similar rates and by the same mechanism and MAS NMR spectra of both forms demonstrated that excellent spectral resolution is maintained with both AβM01-42 and Aβ1-42 and that the chemical shifts are virtually identical in dipolar recoupling experiments that provide information about rigid residues. 3) We developed a novel 3D MAS NMR experiment utilizing through-space 15N-15N correlations. This 3D NNC MAS NMR experiment generates 15N-15N internuclear contacts in protein systems using an optimized 15N-15N proton assisted recoupling (PAR) mixing period and a 13C dimension for improved resolution. The 3D NNC MAS experiment provides an alternate assignment strategy that is complementary to current schemes and, therefore, provides a powerful platform for solid-state protein studies and is broadly applicable to a variety of systems and experimental conditions.
Publications
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A 3D MAS NMR experiment utilizing through-space 15N-15N correlations; J. Am. Chem. Soc. 139 (19), 6518–6521
Donovan KJ, Silvers R, Linse S, Griffin RG
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Aggregation and Fibril Structure of AβM01-42 and Aβ1-42; Biochemistry 56 (36), 4850–4859
Silvers R, Colvin MT, Fredrick KK, Jacavone AC, Lindquist SL, Linse S, Griffin RG
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Atomic Resolution Structure of Monomorphic Aβ42 Amyloid Fibrils; J. Am. Chem. Soc. 138 (30), 9663–9674
Colvin MT, Silvers R, Zhe Ni Q, Can TV, Sergeyev I, Rosay M, Donovan KJ, Michael B, Wall J, Linse S, Griffin RG
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Lipid- Bilayer-Bound Conformation of an Integral Membrane Beta Barrel Protein by Multidimensional MAS NMR; J. Biomol. NMR 61 (3-4) 299-310
Eddy M, Su Y, Silvers R, Andreas L, Clark L, Wagner G, Pintacuda G, Emsley L, Griffin RG
